The present invention relates to a component of bromelain. In particular, the invention relates to the use of this bromelain component in medicine, particularly as an anti-cancer agent and an immunosuppressive agent.
Stem bromelain (bromelain) is the collective name for the proteolytic enzymes found in the tissues of the plant Bromeliaceae. It is a mixture of various moieties derived from the stem of the pineapple plant (Ananas comosus). Bromelain is known to contain at least five proteolytic enzymes but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it may also contain amylase and cellulase activity. In addition, various other components are present.
Bromelain has previously been used in the treatment of a variety of conditions including inflammation and, in particular, it has been used in the treatment of diarrhoea. The use of bromelain in the treatment of infectious diarrhoea is described in WO-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in WO-A-8801506, which teaches that bromelain detaches pathogens from intestinal receptors.
Taussig et al., Planta Medica, 1985, 538-539 and Maurer et al., Planta Medica, 1988, 377-381 both suggest that bromelain may be of use in inhibiting tumour growth. U.S. Pat. No. 5,223,406, DE-A-4302060 and JP-A-59225122 also teach the use of bromelain in the treatment of cancer. U.S. Pat. No. 5,223,406 teaches that bromelain is capable of inducing tumour necrosis factor (TNF) while DE-A-4302060 teaches that bromelain can prevent metastasis by the structural modification of the tumour surface protein CD44.
In WO-A-9400147, various experiments were described which demonstrate that proteolytic enzymes and, in particular, bromelain, are capable of inhibiting secretion. The application also discloses that bromelain can reduce toxin binding activity and can inhibit the secretory effect of toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as heat stable toxin (ST). This is in spite of the fact that ST has a very different mode of action from LT and CT. These observations were explained by the fact that one component of the bromelain mixture, stem bromelain protease, appears to be capable of modulating cyclic nucleotide pathways and this is discussed further in WO-A-9500169. In addition, bromelain has also been demonstrated to inhibit secretion caused by the calcium dependent pathway.
The present inventors have studied the varied biological effects of bromelain and, in particular, its effects in a well documented model of intracellular signal transduction, namely T cell receptor (TCR)/CD3 signalling and IL-2 production. Significant progress over recent years has led to the understanding of biochemical events which occur following TCR engagement (reviewed Cantrell, Annu. Rev. Immunol. 14, 259-274, (1996)), therefore TCR signalling provides an excellent model for elucidation of the effects of biologically active compounds. Effective T cell activation requires two signals. The first signal is generated by the TCR/CD3 complex after engagement with antigen peptide presented by the major histocompatibility complex (MHC) expressed on antigen presenting cells (APC) (Cantrell, 1996). The second, costimulatory signal is generated by ligation of CD28 receptors on T cells with the B7 family of ligands on APC. A key element in the signalling pathway involved in transducing receptor-initiated signals to the nucleus is the family of mitogen-activated protein kinases (MAPk). The best studied of these kinases are the extracellular signal-regulated protein kinases (ERK)-1 and ERK-2 (also referred to as p44MAPk and p42MAPk, respectively). ERK's are serine/threonine kinases that are activated when phosphorylated on tyrosine and threonine residues. In vitro, this activation is reversed if either residue is dephosphorylated. A relatively newly discovered member of the MAPk family are c-Jun NH2-terminal kinases (JNKs) which exist as 46 kDa and 55 kDa forms that also require phosphorylation for activation. ERK activation is dependent on p561Lck and coupling of the TCR/CD3 complex to p21Ras, with subsequent activation of the Raf-1/MEK1/ERK kinase cascade. JNK activation also requires p21Ras, as well as signals generated by the CD28 costimulatory receptor which activate GTP (guanosine triphosphate)-binding proteins (such as Rac1 or Cdc42) that induce the PAK/MEKK/SEK/JNK kinase cascade. Activated ERK phosphorylates Elk-1, which in turn, mediates induction of c-fos activity following phosphorylation of c-jun by JNK. Activated c-fos and c-jun combine to form the AP-1 protein required for IL-2 synthesis. The above events are summarised in FIG. 1. All the above-mentioned signalling events require tyrosine phosphorylation, as inhibitors of protein tyrosine kinases (PTKs) inhibit many events associated with TCR stimulation, including T cell activation and IL-2 production.
In WO-A-9600082, we showed that bromelain could inhibit tyrosine phosphorylation and activation of ERK-2 in T cells stimulated via the TCR, or with combined phorbol ester plus calcium ionophore. We have now found that, in association with decreased ERK activity, bromelain decreased IL-2, IL-4 and IFN-γ mRNA accumulation in T cells stimulated with phorbol ester and ionophore, but did not affect cytokine mRNA accumulation in cells stimulated via the TCR. This data suggests the existence of a TCR-activated, ERK-independent pathway involved in cytokine production in T cells.
From the prior art, it is clear that bromelain is a mixture which has a variety of different physiological effects. Not all of the components of the bromelain mixture have been characterised and so, except for stem bromelain protease, whose activity we have described, it is not clear which of the components is responsible for which of the various different effects of bromelain. This is, of course, a major disadvantage if the bromelain mixture is to be administered as a pharmaceutical because while one component of bromelain might give the desired effect, there may well be unwanted side effects arising from the action of some other component of the bromelain mixture.
It would therefore be beneficial if individual components of bromelain giving rise to particular medicinal activities could be isolated and administered separately so as to lessen the possibility of side effects. We have now identified an active fraction of crude bromelain which is responsible for its ability to inhibit ERK activation, and therefore block the MAP kinase pathway. Although not a single protein, this fraction consists of only a few components and so the possibility of side effects when it is administered to patients is greatly reduced compared with crude bromelain.